Breast Cancer (BC) is one of the most common and challenging cancers among females worldwide. Conventional treatments for oral cancer rely on the use of radiology and surgery accompanied by chemotherapy. Chemotherapy presents many side effects, and the cells often develop resistance to this chemotherapy. It will be urgent to adopt alternative or complementary treatment strategies that are new and more effective without these negative effects to improve the well-being of patients. A substantial number of epidemiological and experimental studies reported that many compounds are derived from natural products such as curcumin and their analogs, which have a great deal of beneficial anti-BC activity by inducing apoptosis, inhibiting cell proliferation, migration, and metastasis, modulating cancer-related pathways, and sensitizing cells to radiotherapy and chemotherapy. In the present study, we investigated the effect of the curcumin-analog PAC on DNA repair pathways in MCF-7 and MDA-MB-231 human breast-cancer cell lines. These pathways are crucial for genome maintenance and cancer prevention. MCF-7 and MDA-MB-231 cells were exposed to PAC at 10 µM. MTT and LDH assays were conducted to evaluate the effects of PAC on cell proliferation and cytotoxicity. Apoptosis was assessed in breast cancer cell lines using flow cytometry with annexin/Pi assay. The expression of proapoptotic and antiapoptotic genes was determined by RT-PCR to see if PAC is active in programming cell death. Additionally, DNA repair signaling pathways were analyzed by PCR arrays focusing on genes being related and confirmed by quantitative PCR. PAC significantly inhibited breast-cancer cell proliferation in a time-dependent manner, more on MDA-MB-231 triple-negative breast cancer cells. The flow cytometry results showed an increase in apoptotic activity. These data have been established by the gene expression and indicate that PAC-induced apoptosis by an increased Bax and decreased Bcl-2 expression. Moreover, PAC affected multiple genes involved in the DNA repair pathways occurring in both cell lines (MCF-7 and MDA-MB231). In addition, our results suggest that PAC upregulated more than twice 16 genes  in MDA-MB-231, 6 genes  in MCF-7, and 4 genes  in the two cell lines. In silico analysis of gene–gene interaction shows that there are common genes between MCF-7 and MDA-MB-321 having direct and indirect effects, among them via coexpression, genetic interactions, pathways, predicted and physical interactions, and shared protein domains with predicted associated genes indicating they are more likely to be functionally related. Our data show that PAC increases involvement of multiple genes in a DNA repair pathway, this certainly can open a new perspective in breast-cancer treatment. Breast cancer (BC) is a major global public health problem being multifactorial and caused by genetic alteration and environmental factors that lead to 2.2 million incident cases and approximately 685 thousand deaths in women worldwide, in 2020. It is now a fatal disease and is considered the second most frequently diagnosed cancer . The conIn response to DNA damage, a series of signaling cascades is activated to promote cell survival. The impact of DNA repair pathways on cancer biology and therapy was documented and considered a key regulator of tumorigenesis . SystemsDifferences in cell morphology were observed between the PAC-treated cells and the controls (with DMSO treatment) by light microscopy. As demonstrated in Morphological changes were more visible in MDA-MB-321 than MCF-7, probably characteristic of breast-cancer cell death and apoptosis. They were more remarkable when the cells were under long-term treatment with PAC or with concentrations > 10 μM.The antiproliferative activity of PAC was evaluated by MTT assay after 24 h in the treatment of MCF-7 and MDA-MB-231. We observed that in both, there was a clear trend of decreasing cell viability in those treated while an increase in proliferation was present among the controls, with significant differences between the two conditions, as illustrated in PAC was substantially suppressing the proliferation at a rate of 60% in MDA-MB-231 and 30% in MCF-7 cells A,C. ThesIn this study, the curcumin analog PAC was screened for its cytotoxic activities against BC cell lines MDA-MB-231 (TNBC) and MCF-7 (HR+ HER2-). LDH assay was performed to assess its cytotoxicity. It significantly caused an increase in LDH release in both after 24 h of treatment in comparison to the vehicle control. Our results shown in An increase in the migratory ability is one thing about a tumor cell that plays a critical role in its metastasis. Therefore, we examined the effect of PAC on motility in breast-cancer cell lines by a wound-healing assay for MCF-7 and MDA-MB-231. As shown in To assess the apoptotic activity of PAC, the annexin V PI test was performed, and the cells were analyzed by flow cytometry. To validate the apoptotic status and possible molecular mechanism of PAC-induced cell death, the apoptosis-related genes (Bax and Bcl-2) were quantified at the mRNA level using RT-PCR analysis. Their degree of expression was assessed in MDA-MB-231 as well as in MCF-7 compared to controls (DMSO). The gene expression was normalized to GADPH. Our results suggest that the expression of the proapoptotic gene (Bax) in both cell lines was increased after treatment with 10 µM of PAC. Versus the vehicle control, our study also demonstrates that the antiapoptotic gene (Bcl-2) had a highly significant decrease after being treated with PAC. Consequently, PAC elevated the Bax/Bcl-2 ratio three times in MDA-MB-231 and twice in MCF-7, as shown in Overall, these findings indicate that PAC promoted apoptosis through the upregulation of proapoptotic genes and the downregulation of antiapoptotic genes.2 Profiler PCR Array  to investigate all interactions between these genes. We selected five query genes (black circles); genes from the PCR array, they correspond to the genes of interest, along the interaction network of all that are connected. This network analysis of the common genes showed direct and indirect interactions among them via coexpression, genetic interactions, pathways, predicted and physical interactions, and shared protein domains with predicted associated genes (gray circles) indicating those that are more likely to be functionally related. Presented in In the current study, we examined the anticancer effects of PAC, a new curcumin analog, on breast cancer cells, especially those in the DNA repair system. It was reported that DNA damage causes cancer development, when erroneous repair is preventing it, particularly the mutations, and by activating oncogenes or inactivating tumor suppressor genes .Firstly, we have found that PAC demonstrated stronger anticancer properties with a more potent effect on ER-negative cells by triggering apoptosis. In addition, the proapoptotic effect of PAC was sixfold higher against ER − (MDA-MB-231) than against ER + (MCF-7). This induction of apoptosis is mediated in the two breast-cancer cell lines through the mitochondrial pathway via the downregulation of Bcl-2/Bax. This outcome comes after that of Al-Hujaily et al. 2011) [ [11]. ThNormal cells are characterized by their ability to preserve genome stability and integrity through capabilities to correct the impaired DNA that can contribute to carcinogenesis ,23. NumeSMUG1 is known to remove uracil and certain oxidized bases from DNA during base excision repair (BER). MUG1 may play a role in preserving genome stability. Kemmerich K et al. (2012) reported that mice carrying a loss of SMUG1 and UNG in combination with loss of mismatch repair (Msh2 −/−) had shortened life spans and increased tumor formation, which implies a role for SMUG1 in protecting cells from the genomic instability that induces cancer, perhaps through direct removal of hmdU moieties . The secIn humans, NER is considered the most versatile DNA repair system able to fix a variety of large lesions, including ultraviolet light-induced DNA photoproducts . The conThirdly, our results demonstrate that PAC induces only one gene involved in nonhomologous end-joining repair; it is named XRCC6 binding protein 1 (XRCC6BP1) in triple-negative breast-cancer cells. Some studies point to the potential of targeting components of the DNA repair pathway, particularly XRCC6BP1, in chemo-resistant lung cancer. Furthermore, they concluded that it may play an important role in lung-cancer stem-cell subsets, at least in part; it may be responsible for chemotherapy resistance . Five otFinally, our results show that one gene in the mismatch repair pathway (three prime repair exonuclease 1 = TREX1) was induced by PAC in triple-negative breast-cancer cells. Lots of studies have found that, in cancer therapy, the dose-dependent efficacy of radiotherapy is at least partially attributable to TREX1 activity . Our bio2 Profiler PCR Array Human DNA Repair (PAHS-042Z) were from Qiagen . The High-Capacity cDNA Reverse Transcription Kit was from Applied Biosystems , and the SYBR Green Master Mix was from Bio-Rad Laboratories, Inc. .The MCF-7 and MDA-MB-231 human breast-cancer cell lines were obtained from ATCC Company . DMEM (Dulbecco’s modified Eagle’s medium), FBS , and antibiotics, P/S (penicillin/streptomycin), were from Thermo Fisher Scientific . As well, Lab-Tek chamber slides were purchased from Nalge Nunc, Inc. . MTT solution, DMSO (dimethyl sulfoxide), trypan blue, and Triton X-100 were received from Sigma-Aldrich . PAC was provided by Dr. Basem Al-Otaibi and Dr. Ibrahim Al-Jammaz from King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. It was diluted in DMSO to reach a 10 mM stock solution and used at c. LDH-Cytotoxicity Colorimetric Assay Kit II was also needed . Annexin V-FITC/Propidium Iodide Kit was from BD Biosciences . RNA Extraction Kit and RT2 cell culture flasks. These were kept in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C. The medium was changed every two days until 90% confluent.The breast-cancer cells were grown in DMEM with the addition of 10% FBS and 1% P/S, in 75 cmMCF-7 and MDA-MB-231 cells were seeded in 2-well Lab-Tek chamber slides with DMEM growth medium. After 24 h adhesion, the cells in one of the wells were treated with 10 μM PAC. Control cells in the second chamber were incubated with the same volume of DMSO-containing medium. After being treated for 24 h, the slides were observed under a phase-contrast microscope and photographed.5 cells/well, in 1000 µL of medium containing 10% FBS. The plates were incubated for one day at 37 °C under 5% CO2 in a humid atmosphere. Then, the cells were treated with PAC (10 µM) or DMSO (vehicle) for 24 h in a growth medium freshly prepared. Following this, the latter was removed after 24-h exposure to the drugs, and a fresh medium supplemented with 5 mg/mL MTT solution was added to each well and put in an incubator for three hours. At the end of this culture period, the supernatant was discarded and 500 µL of 0.04 N HCl in isopropanol was in turn used. Finally, the reaction product was quantified by measuring the absorbance with an xMark microplate reader from Bio-Rad at 550 nM. The proliferation percentage was evaluated in comparison with cells treated by DMSO. This experiment was repeated eight times.The cells were placed in 6-well plates at the density of 3 × 10The cytotoxic effect of PAC was calorimetrically determined with the LDH-Cytotoxicity Colorimetric Assay Kit II. Briefly, cells were treated as described above for the MTT assay and 100 µL of the supernatant was transferred into a 96-well plate according to the manufacturer’s instructions. After that, 100 µL of the reaction mixture was added and the plate was incubated at room temperature in the dark for 30 min. The reaction was stopped by adding 10 µL of stop solution. Finally, LDH release was quantified by measuring absorbance at 450 nm, the reference wavelength was 650 nm using an xMark plate reader. This experiment was done eight times.2 in the humidified atmosphere. The migrating cells were quantified with wound closure metrics, by manually tracing the gap area in captured images, and the migration rate was expressed as a percentage of wound contraction. The photographs were analyzed by using ImageJ software.A wound-healing assay was used as described by our previous works ,17 to ev2 concentration at 37 °C. After 24 h of treatment, the cells were trypsinized, washed twice with PBS, and then stained with Annexin V and propidium iodide, following the manufacturer’s instructions, and next, the apoptotic cells were analyzed with a flow cytometry instrument , as described by our previous works [MCF-7 and MDA-MB-321 cells treated or not with 10 μM PAC, they were afterward incubated for 24 h in a 5% COus works ,67. The Total RNA was extracted by using Qiagen RNA Extraction Kit per the manufacturer’s recommendation. The purity, concentration, and quality of the isolated RNA were all measured with a NanoDrop 8000 from Thermo Fisher Scientific. Next, 1 µg to 2 µg of total RNA was converted into cDNAs using a High-Capacity cDNA Reverse Transcription Kit according to the manufacturer’s directions, as described by our previous works ,68,69.As set out by our previous study , the exp−ΔΔCT). The experiment was repeated four times.Briefly, 12.5 µL SYBR Green, 0.5 µL primer (forward + reverse), 7 µL distilled water, and 5 µL cDNA sample, have been placed in MicroAmp Fast Optical 96-Well Reaction Plates. The plates prepared thereby were run on the 7500 Real-Time PCR System (Applied Biosystems). The PCR samples were preheated at 50 °C for 2 min and at 95 °C for 5 min, followed by 40 cycles . Each reaction has been done in triplicate. By the presence of a single melt peak, the specificity of each primer pair had been verified. The cycle threshold (CT) for each sample was calculated from values using the assisting software from Applied Biosystems. The relative number of mRNA transcripts and the gene-expression quantification value were calculated after normalizing to that of the GAPDH housekeeping gene compared to control samples. Lastly, data were analyzed using the comparative cycle threshold method (22 Profiler PCR Array Human DNA Repair (PAHS-042Z) from Qiagen according to manufacturer instructions [2 Profiler PCR Array is considered the ideal tool for this study. Following the manufacturer’s instructions, after cDNAs were synthesized as described before, for each condition, the components of the PCR mixture were 1350 µL SYBR Green Master Mix, 102 µL cDNA, and 1248 µL RNase-free water; all were put in a 5 mL tube. Next, 25 µL of the PCR mix were added to each well of the RT2 Profiler PCR Array. The plate was centrifuged for 1 min at 1000× g at 25 °C. Then, the real-time PCR was performed. Lastly, relative gene expression was calculated using the 2−ΔΔCT method. The experiment was repeated three times.To determine whether PAC modulates DNA repair pathways, real-time PCR was performed using RTructions ,71. In aA network of all associated genes was used in the study for analyzing gene–gene interactions. To make predictions with their categorized functional protein association implied by genomics, it was determined through GeneMANIA , applied to conduct a network analysis of the common genes and to predict the related ones, it can be installed from the “App Manager” in the Cytoscape software . This network uses the GeneMANIA algorithm and combined data into five groups as follows: coexpression, genetic interactions, pathways, and predicted and physical interactions. GeneMANIA revealed this study that has found an interconnected network of five genes, either through physical interactions, coexpression, prediction, colocalization, pathway, genetic interactions, or shared protein domains that were subjected to gene–gene interaction network analysis using default parameters.−ΔΔCT), and the results were visually represented by fold changes.All experiments were independently repeated at least three times and in triplicate. Data were analyzed with the Statistical Package for the Social Sciences (SPSS) for Windows. They are presented as the mean ± standard deviation (±SD) for continuous variables. For gene expression, the analysis was performed by using the comparative cycle threshold method (2Our data indicate that PAC upregulates multiple genes involved in numerous DNA repair pathways, which certainly can open a new horizon in triple-negative breast-cancer treatment. This compound can be used alone or with other drugs to make breast cancer cells more sensitive to radiation therapy/chemotherapy, and, above all, to reverse the resistance that 80% of these cells develop. Despite this potential, there are currently very few clinical trials testing the many curcumin analogs in combination with chemotherapy or radiotherapy.


Acinetobacter baumannii, an opportunistic pathogen that has rapidly evolved into a superbug exhibiting multidrug-resistant phenotypes, is responsible for a large number of hospital infection outbreaks. With the intensive use of colistin, A. baumannii resistance to colistin has been found to increase significantly. In previous work, we identified a deflazacort derivative, PYED-1 , which exhibits either direct-acting or synergistic activity against Gram-positive and Gram-negative species and Candida spp., including A. baumannii. The aim of this study was to evaluate the antibacterial activity of PYED-1 in combination with colistin against both A. baumannii planktonic and sessile cells. Furthermore, the cytotoxicity of PYED-1 with and without colistin was assessed. Our results show that PYED-1 and colistin can act synergistically to produce a strong antimicrobial effect against multidrug-resistant populations of A. baumannii. Interestingly, our data reveal that PYED-1 is able to restore the efficacy of colistin against all colistin-resistant A. baumannii isolates. This drug combination could achieve a much stronger antimicrobial effect than colistin while using a much smaller dosage of the drugs, additionally eliminating the toxicity and resistance issues associated with the use of colistin.Antibiotic resistance has become more and more widespread over the recent decades, becoming a major global health problem and causing colistin to be increasingly used as an antibiotic of last resort.  Acinetobacter baumannii as one of the six most common and critical nosocomial pathogens  × 100, where Ac represents the OD595 for the control well and At represents the OD595 for the biofilm in the presence of both PYED-1 and colistin. All data points are expressed as means ± SDs of three separate experiments performed in triplicate. The effect of PYED-1 and colistin alone or in combination on the biofilm formation of d method . Brieflysample − APBS)/(ATriton X-100 − APBS), where Asample is the experimental absorbance of PYED-1 and colistin in combination, APBS is the control absorbance of untreated erythrocytes, and ATriton X-100 is the absorbance of 1% Triton X-100-lysed cells. The assays were performed in triplicate and repeated twice.The hemolytic activity of the PYED-1/colistin combination was evaluated by determining hemoglobin release from erythrocytes . Brieflyp < 0.05.Statistical analyses were performed with GraphPad Prism 8 software . All experiments were performed at least three times, and the results are shown as means ± standard deviations (SDs). The significance of the differences was evaluated using one-way ANOVA, followed by Tukey’s multiple comparison test or Dunnett’s multiple comparison test. The differences were considered statistically significant if 1, double bond oxidation with phthalic anhydride in the presence of 50% of aqueous H2O2 gave epoxide 2, in 82% yield, the treatment of which with tributyltin hydride (Bu3SnH) and azobisisobutyronitrile (AIBN) in refluxing tetrahydrofuran (THF) led to the desired PYED-1 in 93% yield  values ranging from 16 µg/mL to 32 µg/mL (with an average of 24 µg/mL), and acted as a bactericidal agent [A. baumannii  on bacterial growth kinetics was separately tested. No differences in A. baumannii (Using checkerboard assays, we first investigated the synergistic action of PYED-1 with colistin against four clinical isolates of colistin-resistant aumannii . A. baumannii strains. The results indicate that PYED-1 potentiated the activity of colistin against all clinical strains . Indeed,In subsequent studies, we evaluated the MBC of colistin in combination with PYED-1 by microdilution checkerboard assays . A. baumannii at concentrations that are ineffective as monotherapy. In recent years, several colistin adjuvant molecules, both antibiotic and non-antibiotic, have been identified to treat infections caused by extensively drug-resistant A. baumannii [In the presence of PYED-1, the MBC/MIC ratio of colistin was less than or equal to 4 for all strains, indicating that the combination colistin/PYED-1 favors killing of aumannii ,36,37,38aumannii ,40. A. baumannii 249 clinical isolate as a model strain. The PYED-1/colistin combination resulted in a strong synergistic effect in A. baumannii 249, which is also a strong biofilm producer.The antimicrobial effect induced by PYED-1 in association with colistin was also evaluated by time–kill studies , using tA. baumannii 249, which was approximately ≥3 log10 lower than the other treated groups already after 6 h of treatment. In agreement with MBC results, time–kill assays confirmed the bactericidal activity of colistin/PYED-1 combination at concentrations that are ineffective as monotherapies.The growth of the tested strain could not be inhibited by either colistin or PYED-1 alone at the concentrations used over 24 h. Conversely, their combination significantly reduced the growth of colistin-resistant S. maltophilia [A. baumannii remains to be elucidated and will be the subject of further investigations.PEYD-1 has been reported to increase membrane permeability in tophilia . AccordiA. baumannii clinical isolates recovered sensitivity to colistin when combined with low concentrations of PYED-1.These results suggest that colistin-resistant A. baumannii lies in its ability to form robust biofilms on healthcare-associated equipment, including central venous catheters, prosthetic heart valves, pacemakers, cerebrospinal fluid shunts and endotracheal tubes [A. baumannii is more tolerant to extracellular stress factors, and higher doses of antibiotics are required to treat its infections involving biofilm formation than in the case of planktonic cells [A. baumannii 249 clinical isolate by crystal violet staining. The results show that PYED-1 and colistin had no effect on biofilm formation of all A. baumannii clinical isolates tested in the range of 256 µg/mL to 2 µg/mL and 2 µg/mL to 0.125 µg/mL, respectively. Then, we carried out biofilm inhibition assays to evaluate the potential synergistic effect of PYED-1 and colistin on the biofilm formed by the A. baumannii 249 strain. Drug concentrations were selected from results determined by the checkerboard analysis. Furthermore, at these concentrations, neither PYED-1 nor colistin could inhibit the bacterial growth. Instead, as shown in A. baumannii when compared with the untreated control and single-drug treatment groups.A main virulence factor of al tubes . Biofilmal tubes . Within ic cells . TherefoA. baumannii biofilm formation [A. baumannii biofilm formation on a polystyrene abiotic surface. Consequently, the use of the combination could prevent or delay both biofilm-related infections and the development and spread of antibiotic resistance in A. baumannii.A few studies demonstrated that treatment with drug combinations inhibits ormation ,43,44,45To look over the therapeutic potential of drug combinations, we assessed the toxicity of the PYED-1 and colistin association. As shown in Significant differences emerged between the experimental groups and the positive control, while there was no significant difference compared to the negative control. Hence, at the synergic concentration, red blood cells did not show a significant hemolytic reaction to the PYED-1/colistin combination.A. baumannii strains below the EUCAST susceptibility breakpoint, thus re-sensitizing the bacteria to the antibiotic drug. Our findings could potentially help clinicians treat infections caused by colistin-resistant A. baumannii. However, further studies are required to investigate the mechanism of action of this fruitful combination of antibiotic agents. Overall, our data indicate that the combination of PYED-1 and colistin displays superior antibacterial activity, as well as no toxicity to mammalian cells. Furthermore, the antibacterial activity of colistin was enhanced more than 256-fold, while PYED-1 was effective at an 8-fold lower MIC value. Importantly, PYED-1 reduced the MIC of colistin against PDR


Gold nanorods (AuNRs) have attracted attention in the field of biomedicine, particularly for their potential as photothermal agents capable of killing tumor cells by photothermic ablation. In this study, the synthesis of novel AuNRs stabilized with thiolated pectin (AuNR@SH-PEC) is reported. To achieve this, thiolated pectin (SH-PEC) was obtained by chemically binding cysteamine motifs to the pectin backbone. The success of the reaction was ascertained using FTIR-ATR. Subsequently, the SH-PEC was used to coat and stabilize the surface of AuNRs (AuNR@SH-PEC). In this context, different concentrations of SH-PEC  were added to 0.50 mL of AuNRs suspended in CTAB, aiming to determine the experimental conditions under which AuNR@SH-PEC maintains stability. The results show that SH-PEC effectively replaced the CTAB adsorbed on the surface of AuNRs, enhancing the stability of AuNRs without affecting their optical properties. Additionally, scanning electron and atomic force microscopy confirmed that SH-PEC is adsorbed into the surface of the AuNRs. Importantly, the dimension size (60 × 15 nm) and the aspect ratio (4:1) remained consistent with those of AuNRs stabilized with CTAB. Then, the photothermal properties of gold nanorods were evaluated by irradiating the aqueous suspension of AuNR@SH-PEC with a CW laser . These results showed that photothermal conversion efficiency is similar to the photothermal conversion observed for AuNR-CTAB. Lastly, the cell viability assays confirmed that the SH-PEC coating enhanced the biocompatibility of AuNR@SH-PEC. Most important, the viability cell assays subjected to laser irradiation in the presence of AuNR@SH-PEC showed a decrease in the cell viability relative to the non-irradiated cells. These results suggest that AuNRs stabilized with thiolated pectin can potentially be exploited in the implementation of photothermal therapy. Cancer stands as a significant global health challenge, primarily attributed to its widespread prevalence, incidence, and high mortality rates. According to the World Health Organization (WHO), in the year 2020, there were 32.74 million individuals afflicted by cancer, with 12.36 million new cases diagnosed, resulting in 6.31 million deaths attributed to this condition in the same year (GLOBOCAN). Traditional cancer treatments encompass surgery, radiotherapy, and chemotherapy. These strategies consist of eradicating the tumor cells and controlling the exacerbated cell growth in the affected tissue or organ and avoiding their dissemination of cancer cells to another body parts ,6,7.The optical properties of gold nanoparticles (AuNPs) have captured the interest of the biomedical field, as they offer valuable potential for the creation of innovative and promising techniques for imaging, diagnostics, and therapy . With th4), Silver Nitrate (AgNO3), Ascorbic Acid and Sodium Tetrahydroborate (NaBH4) were obtained from Sigma Aldrich, and they were used as received.The PEC (74% D-glucoronic acid), Cysteamine Hydrochloride, N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDAC), N-Hydroxysuccinimide (NHS), N, N-Dimethylformamide (DMF), Hexadecyltrimethylammonium bromide (CTAB), Hydrogen -Tetrachloroaurate (III)  and under gently magnetic stirring (100 RPM) using a pH meter GLP 22  .Thiolated pectin (SH-PEC) was synthesized by amidation reaction between the carboxylic acids of PEC with the amino group of cysteamine . Briefly−1) were obtained in ATR mode using a Perkin Elmer Spectrum Two Instrument , at a resolution of 2 cm−1.To ensure the success of the amidation reaction to get thiolated PEC (SH-PEC), FTIR-ATR spectra (400–4000 cmDS) of PEC by cysteamine was determined using 1H NMR spectroscopy. 1H NMR spectra was recorded using a Bruker DRX-500 spectrometer . For the analysis, both PEC and SH-PEC solutions were prepared at a concentration of 1 mg/mL using D2O as a solvent. The NMR spectra were recorded at 300 K, using tetramethyl silane as reference. The DS of SH-PEC was calculated by comparing the signal area of the H1′ of cysteamine (3.40 ppm) to the signal area of H4 (4.40 ppm) corresponding to the galacturonic acid residues [The degree of substitution (residues :DS=H1′3.4 (10 mM) to 5.0 mL of an aqueous solution of CTAB (0.1 M) and HAuCl4 (0.48 mM). The solution was vigorously stirred for 2 min and then kept undisturbed at 28 °C for 30 min. On the other hand, 100 mL of AuNR growth solution was prepared by mixing CTAB, HAuCl4, AgNO3, HCl, and ascorbic acid at final concentrations of 0.1 M, 0.5 mM, 0.085 mM, 0.08 M, respectively, and mixed for 5 min at 500 rpm. Thereafter, 100 μL of Au seeds solution were added to the growth solution, the glass bottle was inverted two times, and then, the solution was kept without perturbation. The color of this solution slowly changed to wine-red color over a period of 3 h. Then, the particle solution was centrifuged  and redispersed in 100 mL of a 10 mM CTAB solution.AuNRs were synthesized by the seed-mediated method . All glaBriefly, 10 mL of AuNRs was washed by centrifugation to eliminate the excess of CTAB and was suspended in 1 mL  of water. Then 0.5 mL of AuNRs solution was added to 4.0 mL of SH-PEC solution , and the final mixture was stirred for 4 h. Finally, the SH-PEC-coated AuNRs (AuNR@SH-PEC) were centrifuged three times to ensure the removal of CTAB  and redispersed in 4.5 mL of water.UV-Vis spectra were obtained by using a spectrophotometer Agilent 8453 scanning from 400 to 1000 nm. The morphological characterization was conducted using both scanning transmission electron microscopy  and atomic force microscopy . STEM analyses were prepared applying a drop of the AuNR@SH-PEC onto carbon-coated copper grids and dried overnight. For AFM analysis, a drop of the AuNR@SH-PEC was applied onto a freshly cleaved mica and left to dry; images were obtained in the noncontact mode using silicon nitride cantilevers . Particle sizes and zeta potential of AuNR@SH-PEC were measured using Zetasizer-Nano Zs .v/v)) and 0.1 mL HNO3 (70% (v/v)). Solutions were diluted with deionized water until reaching a final volume of 2 mL. The intensity of the emission wavelength was measured and compared to a standard solution.The gold concentration in solution was determined by inductively coupling plasma mass spectroscopy (ICP-MS) in a Varian 820-MS equipment , following a previously reported protocol . Briefly2). The temperature rise was measured using a k type thermocouple connected to a digital thermometer, AMPROBE TMD-51. The samples were irradiated for periods of 30 min of turn-on laser and 30 min of turn-off laser.The photothermal properties of AuNR@SH-PECs were analyzed by light irradiation using a continuous wave fiber coupled diode laser source at 808 nm wavelength (1.0 W/cmη) was determined using a previously described model [η is the photothermal conversion efficiency, h is the heat transfer coefficient, S is the surface area of the system, surrT is the temperature of the surrounding environment (25 °C), P is the power output of the laser irradiation (1 W), A808 is the absorbance of the AuNR@SH-PEC at 808 nm, and Q0 represents the heat dissipated by the quartz cuvette water system, which was determined by adjusting the heating of the system without the AuNRs with the help of the following equation:The photothermal conversion efficiency (ed model :(1)η=hST4 cells/well) and grown for 24 h at an optical confluence of 80–90% under standard culture conditions in 100 μL growth medium. After 24 h incubation at 37 °C, 100 μL of AuNRs (1 × 1011–6.25 × 109 AuNRs/mL) with different SH-PEC concentrations (1.0–8.0 mg/mL SH-PEC) in the corresponding cell culture medium were injected into the wells and incubated for 24 h. AuNRs coated with CTAB were used as control. After the corresponding incubation, the culture medium was discarded, cells were washed with 10 mM PBS (pH 7.4) three times, and a fresh culture medium (100 μL) containing 10 μL of CCK-8 reagent was added to each well. After 2 h, the absorption of the samples was measured at 450 nm with a UV-Vis microplate absorbance reader . Cell viability  was calculated as follows:4 cells/well) and grown for 24 h at an optical confluence of 80–90% under standard culture conditions in 100 μL growth medium. After 24 h incubation, cells were treated with AuNR@SH-PEC at a concentration of 6.25 × 109 AuNRs/mL (which is the concentration at which the AuNR@SH-PEC showed an increase in cell viability). After that, cells were irradiated for 5 min with 808 nm laser. The cytotoxicity was measure after 48 h with the same CCK-8 protocol as before.The cytotoxicity of AuNR@SH-PEC was tested in vitro by means of the CCK-8 cytotoxicity assays. Cervical cancer HeLa and fibroblast Balb/c 3t3 cells were seeded into 96-wells plates (1 × 104 cells/well) and grown for 24 h at an optical confluence of 80–90% under standard culture conditions in 100 μL growth medium. After 24 h incubation at 37 °C, 100 μL of AuNRs (6.25 × 109 AuNRs/mL) with different SH-PEC concentrations (1.0–8.0 mg/mL SH-PEC) in the corresponding cell culture medium were injected into the wells and incubated for 24 h. Afterwards, the culture medium was changed for a fresh one, and cells were irradiated with an 808 nm CW laser at 1.0 for 5 min. Afterwards, the ROS production was analyzed using the corresponding kit following the manufacturer’s instructions using a Fluorostar Omega plate reader . Measurements were made in triplicate.Intracellular ROS generation was determined by the ROS assay kit based on the fluorogenic dye molecule 5(6)-Carboxy-2’,7’-dichlorofluorescein diacetate (DFCA-DA). Cervical cancer HeLa and fibroblasts Balb/c 3t3 cells were seeded into 96-wells plates  was used for statistical analysis and graphing. Quantitative data were expressed as the mean ± standard deviation (SD) of three independent experiments (n = 3). All the statistical analyses were assessed using a + ions previous added to the aqueous medium. Then, the first equivalence point is attained when the excess of H+ ions are neutralized [−4 moles of NaOH were necessary to neutralize the number of -COOH groups present in 0.1 g of PEC. Therefore, the degree of esterification is around to 22% (78% of acid groups), which is close to the data reported by the supplier (≥74% acid groups).PEC was chemically modified through an amidation reaction, where the carboxilic acids of PEC reacted with the amino groups of cysteamine in a theoretical degree substitution of 10%. Prior to the chemical modification, the number of carboxylic acids present in the PEC was determined by a NaOH titration. tralized . In the tralized . In the tralized . The numtralized . Based o−1 attributed to the -OH stretching of carboxylic acid. The peak showed at 1730 cm−1, corresponding to the stretching bond of the -C=O of the methyl esters of the carbonyl groups, and the peak at 1600 cm−1 is assigned to the asymmetric tension of -COO−. On the other hand, the SH-PEC FTIR-ATR spectrum contrasted significantly from that of PEC. A new peak located at 2300 cm−1 was assigned to the -SH groups indicating that cysteamine residues have been chemically attached to the PEC [−1 band in the SH-PEC spectrum shows a small shoulder at 1630 cm−1, indicating the formation of secondary amide groups, a result of the chemical reaction between the -NH2 of the cysteamine and -COOH groups of the cysteamine and PEC [The success of the chemical modification of the PEC molecule with cysteamine was confirmed through FTIR-ATR.  the PEC . Additio and PEC .1H NMR spectra recorded for both PEC (black line) and SH-PEC (red line). The 1H NMR spectrum for native PEC displays a complex set of signals ranging from 3.78 to 3.65 ppm. Signals corresponding to esterified methoxyl groups and the H2 of galacturonic residues appeared at 3.77 and 3.70 ppm, respectively [DS of 6.9%.ectively . The douectively . In contectively . To deteectively  was cons3) was multiplied by the volume of an AuNR, which was determined by the TEM micrograph analysis . Then, the molar mass was calculated by multiplying the mass of a single AuNR by Avogadro’s number (NA = 6.022 × 1023). ICP-MS results indicated a gold concentration of 0.0917 g/L. From this result, the molar concentration of AuNRs determined was 1.12 × 10−9 M (1.12 nM).The concentration of the AuNRs obtained at the end of the synthesis process was determined using inductively coupled plasma mass spectroscopy (ICP-MS). Then, the number of AuNRs was calculated based on the number of gold atoms per AuNR . To asce−9 M) with the aim of determining the optimal concentration of SH-PEC needed to get stable AuNRs. The UV-Vis absorption spectrum in max) of 450 nm corresponding to the TSPR, and a second one located at around 780 nm corresponding to the LSPR. However, spectra recorded for AuNRs stabilized with SH-PEC depicted important changes in TSPR and LSPR at certain SH-PEC concentrations. At 0.25 mg/mL (purple line) and 0.5 mg/mL (dark blue line) of SH-PEC, the absorption intensity diminished for both TSPR and LSPR, and a blue shift in the λmax of LSPR were denoted, suggesting that the structure of AuNR is lost due to the fact that the amount of SH-PEC is too low to cover and stabilize the surface of the nanorods. The AuNR@SH-PEC stabilized with 1.0 mg/mL of SH-PEC (green line) resulted in stability in aqueous media; however, a slight broadening of the LSPR was observed due to the formation of small clusters of AuNR@SH-PEC, suggesting that the SH-PEC amount added to stabilize the AuNR solution was in the threshold to cover the surface of the AuNRs. Interestingly, the absorption spectra recorded for AuNR@SH-PEC stabilized with SH-PEC at concentrations of 2.0 mg/mL (pink line) and 4.0 mg/mL (blue line) showed that the transversal and longitudinal SPRs are localized at the same λmax shown by AuNRs stabilized with CTAB, albeit the absorption intensity diminished due to the biopolymeric coating. Finally, the spectrum recorded for the AuNR@SH-PEC stabilized using 8.0 mg/mL of SH-PEC (red line) shows a decrease in the absorption intensity of both TSPR and LSPR; this could be due to the increased thickness of the polymeric coating on the surface of AuNRs. All these results were corroborated by dynamic light scattering (DLS) and Zeta potential (ζ) experiments.The role of SH-PEC as a stabilizer agent for AuNR was assessed by UV-Vis absorption spectroscopy. In this regard, different amounts of SH-PEC (0.25–8.0 mg/mL) were added to an AuNRs solution , the λmax of the longitudinal SPR remains similar to the AuNRs stabilized with CTAB, supporting the role of SH-PEC as stabilizer agent. Based on UV-Vis spectroscopy and the DLS results, it can be concluded that the physical and optical properties of AuNR@SH-PEC with SH-PEC in the range from 1.0 to 8.0 mg/mL can be adequate to be exploited as photothermal devices.DLS and ζ measurements were used to investigate how SH-PEC coating affects the size and stability of the AuNRs (AuNR@SH-PEC). − and -OH groups present on the chemical structure of SH-PEC, forming a multilayer coating and diminishing the number of -COO− exposed to the surface AuNR@SH-PEC. Based on UV-Vis spectroscopy, DLS and ζ potential results, it can be concluded that the physical and optical properties of AuNR@SH-PEC with SH-PEC in the range from 1.00 to 8.00 mg/mL can be adequate to be exploited as photothermal devices.The ζ potential measurements were used to determine the surface charge of the AuNR@SH-PEC and taken to a parameter to confirm (i) the coupling of the SH-PEC molecules in the AuNRs surface and (ii) to get a parameter of stability. η) of gold nanostructures, the experimental data (ΔT versus time) were fitted to a mathematical model (Equation (1)) used by Roper [To evaluate if the photothermic properties of the AuNRs were affected by the SH-PEC coating, the AuNR@SH-PEC solutions were subjected to three continuous cycles of turn-on and turn-off laser irradiation using a CW 808 nm diode laser . The temperature increase was monitored over time. It is important to note that the AuNR@SH-PEC solutions, when subjected to laser irradiation, consistently reached peak temperatures across all three heat cycles. This behavior suggests that the AuNR@SH-PEC remains stable throughout the heat cycles. The SH-PEC coating, in addition to stabilizing the AuNR@SH-PEC solution, also provides thermal stability to the AuNR, preserving its shape. Likewise, the photothermal conversion efficiency was determined following the model described by Roper et al. . In all by Roper .η values determined for AuNR-CTAB and AuNR@SH-PECs. In accordance with this analysis  showed negligible variations, and moreover, the η value remains practically equal to the η calculated for AuNR-CTAB.analysis , it can ed light . On the 9 to 1.00 × 1011 AuNRs/mL, without subjecting them to laser irradiation. These results are shown in 11). On the other hand, the survival of the Balb/c 3t3 cells incubated in the presence of 6.25 × 109 AuNR@SH-PEC/mL, for all formulations of AuNR@SH-PEC tested, was around 100%, suggesting that the replacing of CTAB for SH-PEC results in a biocompatible nanomaterial. Contrarily, HeLa cells showed greater susceptibility to the presence of AuNR@SH-PEC. As shown in 9 and 1 × 1011 AuNR@SH-PEC/mL). Furthermore, there was a noticeable increase in the toxicity of AuNR@SH-PECs to HeLa as the amount of SH-PEC used for the stabilization of gold nanorods increased. For instance, at a concentration of 6.25 × 109 AuNR@SH-PEC/mL, the HeLa cell survival rate dropped from 97% to 54% when the cells were exposed to AuNR@SH-PEC stabilized with 8.0 mg/mL within the concentration range of 6.25 × 109 to 1 × 1011. Based on these results, the concentration of 6.25 × 109 of AuNR@SH-PEC/mL was chosen to assess the photothermal effects on the viability of both Balb/c 3t3 and HeLa cells. This decision was made to avoid potential toxicity arising from the type and concentration of AuNR@SH-PEC.The photothermal effect of AuNR@SH-PECs on the viability of Balb/c 3t3 and HeLa and was evaluated by in vitro assays. The cytotoxic activity of AuNR@SH-PEC was assessed by conducting tests involving AuNR-CTAB and AuNR@SH-PECs at concentrations ranging from 6.25 × 109 AuNR@SH-PEC/mL, irradiating with a CW laser . As depicted in Then, the photothermal effect of gold nanorods on the viability of Balb/c 3t3 and Hela cells was assessed using a concentration of 6.25 × 10Reactive oxygen species (ROS) play a pivotal role in various cellular functions, including differentiation and the killing of cancerous cells ,56. In tIn summary, our research culminated in the successful development of a novel nanoplatform, AuNR@SH-PEC. In accordance with the results, the methodology used to produce this nanomaterial is easy to develop and is replicable. Initially, a chemical modification of PEC was implemented to attach cysteamine molecules to the biopolymer structure and to introduce -SH groups, which have a high affinity for gold surfaces. This facilitated the substitution of CTAB, initially adsorbed onto the surface of AuNR, with SH-PEC molecules, resulting in a stable gold-based nanosystem. Importantly, the optical and photothermal properties of the synthesized AuNR@SH-PECs are comparable to those observed in AuNR-CTAB. Notably, the in vitro assays showed that AuNR@SH-PEC has superior biocompatibility compared to AuNR-CTAB. This consequently enhances its suitability for effortless integration into biological systems. The viability assays confirmed the potential of AuNRs@SH-PECs as a photothermal agent since the survival of HeLa cells was reduced to 40%. Based on these data, it is confidently stated that these nanoplatforms show significant potential for advancing the field of photothermal therapy, introducing a novel option in the fight against formidable diseases such as cancer.


BackgroundWe aimed to analyze the expression of infection-related biomarkers and inflammatory cytokines in laboratory-confirmed cases and compare the differences between clinically severe and non-severe ones.MethodWe randomly selected 35 patients who were hospitalized with the diagnosis of coronavirus disease 2019 (COVID-19). Blood serum was obtained at the time of admission to the hospital, on the third to the fifth day, and at the time of discharge.ResultThe median age of our patients was 56.5±69.7 months (range: 1-205 months). The mean pro-B-type natriuretic peptide (pro-BNP) was significantly higher at the time of admission than on the third to the fifth day of illness. The mean pro-B-type natriuretic peptide levels at three time points were significantly higher in patients with severe cases than in mild-moderate cases. However, there was no significant difference between the clinical severity with regard to the cytokine levels at disease onset and recovery.ConclusionIn the study, it was shown that cytokines play an important role in the pathogenesis of COVID-19. Therefore, it may be beneficial to use agents such as tocilizumab in the treatment. Coronavirus disease 2019 (COVID-19) has become a public health threat to people all over the world, which was declared a pandemic on March 11, 2020. Globally, as reported by the World Health Organization (WHO) on March 21, 2023, there have been 761,071,826 confirmed cases of COVID-19, including 6,879,677 deaths (https://covid19.who.int/). The lower airway tract is the primary target of the infection ,5. The rThis prospective study aimed to analyze the expression of infection-related biomarkers and inflammatory cytokines  in laboratory-confirmed cases and compare the differences between clinically severe and non-severe ones.We randomly selected 35 patients who were hospitalized with the diagnosis of COVID-19 at the local state and university hospitals during the period between May 25, 2020, and August 12, 2020. Written informed consent was obtained from the parents. Data including demographic characteristics, medical history, symptoms, signs, and laboratory findings were collected from the patients’ medical records. Blood serum was obtained to study routine blood tests, infection-related biomarkers, and inflammatory cytokines  at the time of admission to the hospital, on the third to the fifth day, and at the time of discharge. Serum TNF-α , IL-1β , and IL-6  levels were measured using the enzyme-linked immunosorbent assay (ELISA) method. The test results were expressed in pg/mL. The methods were applied in accordance with the protocols provided by the manufacturers of the kits. Measurement was performed using the Multiskan FC photometric microplate reader . The severity of COVID-19 was determined according to the Fifth Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment Guidance [Statistical analysisWe described the categorical variables as frequency rates and percentages, and continuous variables as mean and standard deviation (SD). The T-test and the Mann-Whitney U test were performed for two-group comparisons. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 20.0 software . Two-sided P-values of less than 0.05 was considered statistically significant. Correlation between laboratory variables and cytokines was sought using a nonparametric test of correlation (Spearman’s rank correlation test).The median age of 35 patients in total was 56.5±69.7 months (range: 1-205 months); 18 (51.4%) patients were male. Seventeen (48.6 %) patients had a history of contact with a COVID-19-positive patient. None of the patients received SARS-CoV-2 vaccination. The presenting symptoms were mostly associated with one another, which included fever , cough , respiratory distress , nasal congestion/runny nose , headache , myalgia , and sore throat . One patient had also abdominal pain. The physical examination findings were as follows: 18 (51.4%) patients had rales/rhonchi on auscultation of the lungs, four (11.4%) patients had cyanosis at hospital admission, 13 (37.1%) patients had wheezing, 10 (28.6%) patients had signs of respiratory disorders , and 16 (45.7%) patients had tachycardia and tachypnea. Two patients were asymptomatic. Ten (28.6%) patients’ clinical presentation was severe, and they were followed in the intensive care unit (ICU). The remaining 23 patients’ clinical severity was mild-moderate. One (2.9%) patient who had a congenital heart defect (transposition of the great arteries (TGA)) died because of the disease. Two (5.7%) patients had cerebral palsy and epilepsy. These patients were intubated and were followed under mechanical ventilation.The mean leucocyte, neutrophil count, serum pro-B-type natriuretic peptide (pro-BNP), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α serum levels were significantly higher at the time of admission than recovery. The mean eosinophil count, mean corpuscular volume (MCV), C-reactive protein (CRP), creatinine kinase (CK), ferritin, aspartate transaminase (AST), and alanine transaminase (ALT) levels were significantly lower at the time of admission. The mean D-dimer, CK level, and ferritin levels were significantly higher at peak disease than at the time of admission. The mean pro-BNP was significantly higher at the time of admission than at the disease peak. Moreover, the pro-BNP level of the patient who died was relatively very high . Coronavirus PCR was positive in 31 (88.6%) patients at the time of admission, while it became positive at the disease peak in the remaining four (11.4%) patients. After the disease was cured, the PCR became negative in all patients, except the patients who died because of the disease . However, this difference was statistically nonsignificant. The mean neutrophil count and CRP on the third to the fifth day, the mean pro-BNP level at three time points, the mean CK level at the disease peak and the end of the disease, and the mean ferritin and D-dimer levels on the third to the fifth day were significantly higher in patients with severe clinical presentation. However, there was no significant difference between the clinical severity with regard to the cytokine levels at the time of admission and recovery Table .Six (17.1%) patients had widespread ground-glass opacity (GGO) and multifocal consolidation appearance on the thoracic computed tomography (CT). Two (5.7%) patients had minimal GGO on CT. Eleven 31.4%) patients took bronchodilators ; 33 (91.4%) patients additionally took different antibiotics including azithromycin. None of the patients received tocilizumab. Nine (25%) patients received steroids. Seven (20%) patients took oseltamivir; six (17.1%) patients received Kaletra (lopinavir/ritonavir). Four (11.4%) patients took favipiravir; seven (20%) patients additionally took hydroxychloroquine. Three (8.6%) patients needed nasal continuous positive airway pressure (CPAP) application. Two (5.7%) patients needed mechanical ventilation. One (2.9%) patient died because of COVID-19. The mean duration of COVID-19 PCR positivity was 6.7±2.2 days , which was similar to the figure reported in our study . Our youOur short-time experience with COVID-19 showed that symptoms are less severe in children than in adults . PreviouIn children, the severity increased as the patient’s age decreased. A study reported by Zhen-Dong et al.  showed tPro-BNP was highest at the time of admission to the hospital. It was also remarkably higher in severe patients and again at the time of admission. This suggests that pro-BNP may be an adverse prognostic factor for COVID-19. Our results are consistent with a previous adult study that showed that NT-pro-BNP might be an independent risk factor for in-hospital death in patients with severe COVID-19. That study enrolled 102 adult patients with severe COVID-19 . PreviouOur results related to complete blood count parameters are inconsistent with other studies that have shown low proportions of neutrophilia (4.6%), neutropenia (6%), and lymphocytopenia (3%) -22. Our Chen et al. (2020)  found thOne of our patients who was one month old died. She had an underlying disease, namely, the transposition of great arteries (TGA). The COVID-19 test was positive at the onset and peak of the disease. Her pro-BNP was relatively very high at the time of admission. Her clinical course became more severe in hours, so there was no time for taking a CT. However, Guo et al. (2020)  showed tAccording to Lu et al. , GGO wasCOVID-19 can be detected in different body specimens. A study with 205 patients showed that nasal swab positivity was 63% and pharyngeal swab positivity was 32% . In our Most reports have suggested supportive treatment, including oxygen therapy and antibiotics for bacterial superinfections . AntivirOur study has some limitations. First, we did not evaluate the cytokine levels at peak disease. Second, the patient group is not homogenous since there are more severe patients than those included in previous studies. Third, this study only included a small number of patients; thus, the results should be interpreted with caution, and statistical nonsignificance may not rule out differences between severe and moderate cases.In conclusion, our pediatric experience with COVID-19 showed that mortality is relatively low even in the ICU compared to adults. Moreover, cytokines play an important role in the pathogenesis of COVID-19. So, specific treatment options such as tocilizumab (anti-IL-6) may be utilized for clinically critical patients. Cytokines are often elevated in children with COVID-19, but we did not note any association of cytokine levels with disease severity.


To re-explore the responsiveness of the Persian version of Multiple Sclerosis Walking Scale-12 (MSWS-12p) to physiotherapy intervention and determine the minimally clinically important change (MCIC). This study followed a prospective cohort design. Patients with MS (PwMS) underwent physiotherapy treatment for 10 sessions. The outcome measures were the MSWS-12p and Timed 25-Foot Walk test (T25-FW). Data was collected before and after ten sessions of physiotherapy. The effect sizes and the area under receiver operating characteristics curve (AUC) and MCIC were calculated.Thirty PwMS  participated in the study. The effect sizes for MSWS-12p were moderate . The change scores of MSWS-12p showed excellent correlation with the dichotomized smallest detectable change (SDC) criterion (Eta coefficient test = 0.84). There was no correlation between the MSWS-12p total change scores and the T25-FW . The AUC was perfect and the MCIC for the MSWS-12p was calculated 10.0 points. The MSWS-12p is responsive and demonstrates changes after physiotherapy. Changes > 10.0 points on MSWS-12p total score should be considered as true improvement after physiotherapy. Multiple sclerosis (MS) is a prevalent disabling disease of the central nervous system. The prevalence of MS in Iran is 16.5 and 44.8 per 100,000 people in men and women, respectively , 3. WalkThe MSWS-12 is a self-reported questionnaire that is widely used to measure the impact of MS on walking ability and to detect meaningful changes after treatment of PwMS. Most self-reported questionnaires are in English and for use in other languages must be well translated and adapted culturally for the content validity and equivalency with the source version. The adapted version of a questionnaire needs to be examined for other measurement properties through the process of validation one of which is responsiveness. Validity ensures the scores of a questionnaire are consistent with the construct being measured.The responsiveness is the ability of a questionnaire to accurately detect any clinical important change, when even small change is occurred. The responsiveness of a questionnaire indicates that the real important change is distinguished from the change due to measurement error.The MSWS-12 has been adapted and validated into Persian language (MSWS-12p) . A recenPwMS who were referred to Physiotherapy Clinic of Iran MS Society were included in the study. This prospective, pretest-posttest study was conducted from March 2019 to March 2020 in Tehran, Iran.Inclusion criteria were: (1) ability to walk with or without walking aids ; (2) ability to read and write in Persian; (3) ability to follow the commands; and (4) giving consent to participate in the study. Exclusion criteria were: (1) presence of other neurological disorders; (2) not completing the physiotherapy program; and (3) not completing the all questionnaire items.Patients willing to participate in the study were evaluated for eligibility. Demographic and basic characteristics of patients including age, gender, body mass index (BMI), and years since MS diagnosis were recorded. The patients were then asked to complete the MSWS-12p questionnaire. Thereafter, patients were tested using the T25-FW before initiating physiotherapy. Physiotherapy in the form of exercise therapy was presented to the patients, three days per week, and 45 min for each session. The physiotherapy treatment was individualized for each patient and consisted of muscle stretching, general conditioning exercises, walking and balance exercises. At the end of the 10th session of physiotherapy, the participants completed the MSWS-12p questionnaire and were re-tested for the T25-FW.The MSWS-12p is a patient-reported outcome measure that includes 12 items, each item is scored from “1”  to “5” (extremely affected) with the summed score ranging from 12 to 60. The total score from 0 to 80 was calculated [patient’s score – 12 (minimum score possible)/60 (the maximum score) ×100] [.The T25-FW is a measure of walking speed , 13. Thepre – Mpost/SDpre). The standardized response mean (SRM) was calculated by dividing the mean change scores by the SD of change scores. Effect sizes of 0.2, 0.5, and 0.8 were interpreted as small, medium, and large, respectively [The mean and standard deviation (SD) were calculated for quantitative variables. Kolmogorov-Smirnov (KS) test was used to examine the normality of the data. Paired T-tests were used to examine the change before and after physiotherapy. The standardized effect size (SES) was used to calculate the effect size  from “Unimproved” (< 8.8) patients according to the SDC cut-off of 8.8 points for MSWS-12p; Independent-Samples T-Tests was used to examine the difference between two groups on the MSWS-12p and T25-FW mean change scores . The areThirty patients with a mean age of 43.1 years  participated in the study, of whom 16 were female. The mean duration of MS disease was 13.75 years (SD = 6.31). The mean BMI of participants was 23.97 (SD = 4.6).The mean and SD for MSWS-12p and T25-FW with effects sizes are shown in Table ROC analysis of the MSWS-12p showed the AUC = 1.0. The best MCIC for MSWS-12p was 10.0 points (both sensitivity and specificity 100%) to distinguish PwMS who had an improvement in walking from those who had not (Fig. The current study aimed to reevaluate the responsiveness of the MSWS-12p and determine the MSWS-12p MCIC to observe whether the prior findings recur. In this study, both distribution-and anchor-based methods were applied , 19. TheThere was a non-significant correlation between the MSWS-12p and the T25-FW consistent with a previous investigation . This inThe correlation between the MSWS-12p and the SDC dichotomized category was excellent and supports its use in identifying patients who are truly improved from those who are unimproved. The SDC implies that the effect of intervention in PwMS using the MSWS-12p as the outcome measure has to be greater than the 8.8 points .In this study, the SDC based analysis of ROC produced a perfect AUC. As well, the MCIC value of 10 points based on SDC criterion for MSWS-12p found in this study is beyond the measurement error. In line with our finding an MCIC of 10.4 was reported previously . HoweverThe MSWS-12p was found to be responsive when applied in PwMS. Based on the SDC value, the MCIC was calculated as 10 points and is beyond the measurement error of 8.8 points. A change of at least 10 points on the MSWS-12p must occur to be considered as an important clinically meaningful change. Further investigations should confirm our approach and MCIC value.First, the sample size was small. At least 50 PwMS is necessary according to COSMIN guideline , 35. We


Other balance metrics indicative of transfer had lower effect sizes . Given the limited transfer across balance training tasks, future work should examine how intermittent visual occlusions during multi-task training improve real world functional outcomes.The goals of this study were to determine if a single 30-minute session of practice walking on a treadmill mounted balance beam: 1) altered sacral marker movement kinematics during beam walking, and 2) affected measures of balance during treadmill walking and standing balance. Two groups of young, healthy human subjects practiced walking on a treadmill mounted balance beam for thirty minutes. One group trained with intermittent visual occlusions and the other group trained with unperturbed vision. We hypothesized that the subjects would show changes in sacrum movement kinematics after training and that there would be group differences due to larger improvements in beam walking performance by the visual occlusions group. We also investigated if there was any balance transfer from training on the beam to treadmill walking (margin of stability) and to standing static balance (center of pressure excursion). We found significant differences in sacral marker maximal velocity after training for both groups, but no significant differences between the two groups from training. There was limited evidence of balance transfer from beam-walking practice to gait margin of stability for treadmill walking and for single leg standing balance, but not for tandem stance balance. The number of step-offs while walking on a narrow beam had the largest change with training (partial η Practicing a balance task that not only improves performance on that balance task but also transfers improvement to other balance tasks would be ideal for motor skill training and rehabilitation. Recent studies have mixed findings on the transfer of balance training. Many studies that have examined standing balance training have found little to no transfer of balance improvements to other balance tasks –4. In coOne potential training method for challenging dynamic balance is walking on a narrow beam. Studies on humans walking on an overground balance beam have been useful for understanding motor skill expertise –9 and stSuccessful locomotion across a narrow beam requires humans to control movement of their center of mass (CoM) to prevent stepping off the beam. In normal gait, humans actively maintain medio-lateral balance by controlling foot placement for each step –21. WalkBrief intermittent visual perturbations during beam walking practice can improve subsequent beam walking performance , 22. Peta priori predict a direction of the change from pre-test to post-test. We also compared other metrics of balance during treadmill walking and standing balance to determine if there was any evidence of transfer from the balance beam practice to other balance measures.In this study, we sought to identify changes in body movement patterns related to improved beam walking performance with practice, and if there was transfer of better dynamic balance from beam walking to other balance tasks. Specifically, we wanted to determine if a single 30-minute session of practice walking on a treadmill mounted balance beam: 1) altered sacral marker movement kinematics during beam walking, and 2) affected measures of balance during treadmill walking and standing balance. We chose sacral movement as the primary focus of our kinematic analysis for beam walking performance because it is a simple method and good approximation for estimating CoM motion during walking –32 and tTwenty healthy, young participants  took part in this study. They reported no neurological, orthopedic, or musculoskeletal conditions or lower limb surgeries. All participants were right-foot dominant. We assessed foot-dominance by asking them which foot they would use to kick a ball. Written, informed consent was obtained from all study participants and the study was approved by the University of Florida Institutional Review Board (IRB). The study was conducted according to the Declaration of Helsinki.Participants performed a beam walking task with or without the presence of reoccurring intermittent visual occlusions . The balThe visual occlusions were presented in a periodic manner using a Samsung Note 10 that was running Senpatec app . The occlusion cycle would start when participants stepped on the beam with both feet and ended when they stepped off. A random delay of up to 1 s was added to the start of the cycle to avoid any anticipatory effects. To start and stop the cycle we used TeamViewer and a custom Matlab script to access the screen of the phone over a desktop monitor. Every time participants stepped off the beam, they had to count 5 steps before stepping back on –17. The Participants performed the walking and static balance tasks on a split-belt treadmill  that recorded forces through embedded force plates with a sampling rate of 1000 Hz. Participants walked for 5 mins and performed a 30-sec tandem and single leg stance before (pre), and after (post) the beam walking task . The ordMarker trajectories were cleaned using Motive software  and processed with Visual3D . We low-pass filtered the marker data using a 6 Hz cut-off frequency and fourth-order Butterworth filter and determined on-beam phases using a manual threshold for the position of the toe, fifth metatarsal, and heel foot markers in the medio-lateral and vertical directions for each step. We used a manually adjusted, empirically determined threshold because the beam location varied slightly across subjects and sessions. A number of reflective markers (n = 28) were placed on the participants’ feet, knees, hips, sacrum, back, shoulder, neck, and head, to record motion capture data  sampled at 100 Hz for all tasks .We calculated the sacrum kinematics when participants were on the beam in the medio-lateral, anterior-posterior, and vertical direction. We calculated the average standard deviation, as a surrogate for the CoM variability, during the pre- and post- trials for each participant and averaged the values within the two groups. To identify if the visual occlusions group employed faster corrective adjustments after the training compared to the unperturbed vision group, we looked at sacrum peak displacement and velocity for the pre- and post-trials. To do so, we calculated the highest absolute value of displacement and velocity of the sacrum marker for each trial and participant, and then averaged within groups. To investigate if participants adopted a more crouched position after training, we calculated the normalized sacrum-ankle distance for the pre- and post- trials. For each on-the-beam stance phase within each trial, we averaged the vertical component of the sacrum and ankle marker and found the distance between the two. This distance was normalized to the sacrum-ankle distance of the participant’s upright stance when off-the beam. Normalized sacrum-ankle distance values were averaged within trials and groups.We calculated the dynamic margin of stability to see if there was any transfer of the gained balance benefit from the balance beam training to the treadmill walking . We estig is the gravitational constant, and l is the equivalent pendulum length, i.e., the mean distance from the leading foot’s fifth metatarsal marker (left or right) to the center of mass at every heel strike. The dynamic margin of stability (MOS) was then calculated asWhere x is the CoM position, Where BOS is the boundary of the base of support. Dynamic margin of stability and base of support were both measured separately in the anterior-posterior and medio-lateral directions. The base of support was defined by the position of the leading foot marker at heel strike. Dynamic margin of stability values were calculated at heel strike for each step in the pre- and post-walking test and averaged within the whole trail. Heel strikes were detected using the force plates positioned underneath the split-belt treadmill.For the tandem stance and single-leg stance task we low-pass filtered the force plate data using a 12 Hz cut-off frequency and calculated several center of pressure (CoP) measures: i) average CoP sway excursion, ii) Standard Deviation of CoP sway excursion, ii) mean CoP velocity in the medio-lateral and anterior-posterior direction, and iv) area of CoP sway excursion during tandem stance and single-leg stance. For all data analysis we used custom Matlab scripts .We had to exclude one participant from each group from the whole analysis due to missing force plate and marker data during the experiment . We had to exclude an additional participant from each group from the walking and static balance tasks analysis due to missing force plate and marker data during some of the relevant trials . We performed a repeated measures ANOVA with the intervention  as the between subjects’ factor and the test-trial  as the within subjects’ factor for the on-beam measures i) sacrum standard deviation ii) maximum sacrum displacement ii) maximum sacrum velocity in the medio-lateral, vertical, and anterior-posterior direction, and iii) ankle-sacrum ratio. The same statistical analysis with the same between and within subject factors was performed for the beam performance in step-offs/min. Similarly, we performed a repeated measures ANOVA with the intervention  as the between subjects’ factor and the time point  as the within subject’s factor for the i) average CoP sway excursion, ii) SD of CoP sway excursion, ii) mean CoP velocity in the medio-lateral and anterior-posterior direction, and iv) area of CoP sway excursion during tandem stance and single-leg stance. We performed the same statistical analysis with the same between and within subject factors for the i) mean margin of stability, ii) margin of stability variability in the medio-lateral and anterior-posterior direction during walking. Before deciding to use a parametric statistical test for the above measures, we checked the normal distribution of our data. Most of our data was normally distributed. Our primary and secondary outcome measure were step-offs/min and sacral marker standard deviation, which were both normally distributed. We expected limited to no changes in other balance metrics on the beam, and/or during standing and walking. Last, all significant results reported in the next section came from normally distributed data, therefore, violation of normality within some of our measures did not affect our results. We report Greenhouse-Geisser corrected values wherever the test of sphericity is violated.2 = .66). The visual occlusions group showed a four-fold reduction in step-offs  = 14.9, p < .001, partial η2 = .48, t(8) = 4.9, p = .001) while the unperturbed vision group showed 23% improvement between pre-test and post-test (t(8) = 2.9, p = .02) (2) of the test-trial (~0.7) and visual occlusion training (0.5) on step-offs were large  = 31.4, p < .001, partial ηp = .02) . Both efre large .2 = .17), vertical  = .69, p = .42, partial η2 = .04), and anterior-posterior direction = 4.2, p = .06, partial η2 = .2)  = 1.1, p = .31, partial η2 = .07; vertical: F  = .2, p = .18, partial η2 = .11)  = 3.3, p = .092, partial ηη2 = .2) . The tra2 = .11) .2 = .24) and training type  = 8.7, p = .009, partial η2 = .35) on maximal sacral vertical velocity. There was also a significant training type effect on mediolateral maximal sacral velocity  = 5.1, p = .04, partial η2 = .24), as the intermittent visual occlusion group had a decrease and the unperturbed vision group has a very small increase  = 4.9, p = .04, partial ηincrease , S1 Fig.2 = .5), but there was not a statistical difference between groups  = 15.8, p = .001, partial ηn groups , S2 Fig.2 = .27)  = 5.11, p = .04, partial η2 = .27) . There w2 = .50) and anterior-posterior  = 8.8, p = .01, partial η2 = .38) center of pressure velocity  = 14, p = .002, partial ηvelocity . We did velocity , S3 Fig.2 = ~0.7 and η2 = 0.5, respectively). Thirty minutes of treadmill mounted beam walking practice had a large effect on motor performance, and the intermittent visual occlusions had a large effect on boosting the improvement.Both groups showed a significant reduction in step-offs after the beam walking training. The visual occlusions training led to a higher reduction of step-offs (75%) than the unperturbed vision training (23%). Both within subjects  and between subjects (training type) effects sizes were large  compared to step-offs off the beam , supporting the task specificity of balance training principle [We found limited transfer of balance beam performance gains to treadmill walking margin of stability and single-leg stance center of pressure velocity. These measures had lower effect sizes partial η<0.5 comprinciple , 51, whiThere were several limitations to the study that should be considered for future work. The sample size was relatively small. The number of subjects had been chosen based on the expected effect size of the improvements in step-offs. Indeed, we had a large effect size for the training type (~0.5) and for the pre- to post-training (~0.7) in regard to the primary performance metric (beam step-offs). The effect sizes for the sacral kinematic measures that were significantly different after beam practice were equal to or less than 0.35. Future studies that want to determine if there were small changes in sacral or CoM movement dynamics that we did not detect would ned to recruit a larger sample to account for the smaller effect sizes. Another limitation of our study was that we only looked for differences in sacral movement patterns and transfer to other balance tasks after one 30-minute training session. It is possible that many sessions of training over days or weeks would have a larger effect on sacral movement dynamics and transfer to other balance tasks. We chose the time of training for this study based on the expected large effect size from 30 minutes of practice. Symeonidou and Ferris , 23 showOur results support the benefits of treadmill mounted beam walking and intermittent visual occlusions as means to improve dynamic balance in healthy, young humans. There were few indicators of changes in sacral movement dynamics after training that may have been responsible for the large improvements in beam walking performance after half an hour of practice. Balance performance during single leg stance and treadmill walking showed some limited transfer effects after beam walking practice, but the overall transfer was not large. Future studies examining the effects of intermittent visual occlusions using longer training interventions with multiple motor tasks on balance-challenged individuals are warranted.S1 Fig2 = .24). The visual occlusions group also showed a decline in maximum sacrum velocity between pre- and post-test. In the vertical direction we saw a main effect of training type  = 8.7, p = .009, partial η2 = .35) and a main effect of test-trial  = 4.9, p = .04, partial η2 = .24). Error bars represent standard error of the mean.Medio-lateral and vertical sacral marker maximum velocity for the visual occlusions (red) and unperturbed vision group (blue) before (Pre) and after (Post) the training. In the medio-lateral direction there was a significant main effect of training type  = 5.1, p = .04, partial η(TIF)Click here for additional data file.S2 Fig2 = .5), meaning both adapted a more crouched position after training. The visual occlusions group showed ~0.7 cm reduction, while the unperturbed vision group showed a larger deduction of ~1.7 cm. Error bars represent standard error of the mean.Normalized ankle-sacrum distance for the visual occlusions (red) and unperturbed vision group (blue) before (Pre) and after (Post) the training. Both groups showed a significant reduction of ankle-sacrum distance between pre- and post-test  = 15.8, p = .001, partial η(TIF)Click here for additional data file.S3 Fig2 = .50) and anterior-posterior direction  = 8.8, p = .01, partial η2 = .38). Error bars represent standard error of the mean.Mean mediolateral and anterior-posterior center of pressure (CoP) velocity during the single-leg stance task. Both groups showed a decrease in CoP velocity in the medio-lateral and anterior-posterior direction. There was a significant main effect of test-trial in the medio-lateral  = 14, p = .002, partial η(TIF)Click here for additional data file.